A High-Throughput Method for Directed Evolution of NAD(P)⁺-Dependent Dehydrogenases for the Reduction of Biomimetic Nicotinamide Analogues.

ABSTRACT: Engineering flavin-free NAD(P)⁺-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNAD⁺s) is of importance for eliminating the need for costly NAD(P)⁺ in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD⁺ specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD⁺-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD⁺-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat

SUBMITTER: Huang R 

PROVIDER: S-EPMC8580058 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature